Application of OPO and / or OPL in preparation of product for improving anti-stress capability

Abstract

The invention provides an application of OPO and / or OPL in preparation of a product for improving anti-stress capability, and the product specifically improves heat stress resistance and / or ultraviolet stress resistance, and also has the effect of delaying senescence or prolonging life. The invention also provides an application of OPL and OPO in improving the heat stress resistance and / or ultraviolet stress resistance of caenorhabditis elegans. The product has the effect of delaying senescence or prolonging the service life, provides a theoretical basis for further development, utilization and popularization of OPL and OPO, and can be applied to foods or medicines.

Description

Technical Field

[0001] The present invention relates to the field of food or medicine, and specifically to an application of OPO and / or OPL in preparing a product for improving stress resistance, and specifically to an application of OPO and / or OPL in preparing a product for improving heat stress resistance and / or ultraviolet stress resistance. Background Art

[0002] C. elegans is non-toxic, small, has a short life cycle, low food intake, is easy to cultivate, and can survive independently. C. elegans has a clear lipid metabolism network, and fatty acids mainly rely on exogenous food. Its lipid metabolism genes are highly conserved with those of humans. C. elegans is also the first multicellular eukaryote to be sequenced in its entirety. More than 70% of its genes are homologous to human genes. It is a good model animal for growth and development, lipid metabolism regulation, obesity, and the pathogenesis of obesity-related metabolic diseases. The biological information obtained from studying C. elegans is highly adaptable to humans and higher animals.

[0003] Stress is a series of non-specific systemic adaptive reactions that occur when the body is over-stimulated by biological, physical and chemical factors in the external and internal environment. Heat stress is the sum of non-specific responses that occur when a person or animal is stimulated by excessively high temperatures that exceed their body temperature regulation capabilities. Under excessive temperature stimulation, the normal thermal balance is disrupted, and the human body will undergo a series of complex physiological and psychological changes. Whether it is a human or an animal, its body functions will be affected by heat stress.

[0004] Stress resistance refers to the ability to reduce stressor stimulation and enhance the ability to adapt under stress load. It is a self-protection mechanism that individuals show when facing stressors, including both physiological adjustments and psychological adaptation and recovery. Through this mechanism, people can better cope with various challenges and pressures in life, thereby maintaining physical and mental health.

[0005] 1,3-olein-2-palmitintriacylglycerols (OPO) and 1-olein-2-palmitin-3-linoleintriacylglycerols (OPL) are the sn-2 palmitic acid triglycerides with the highest content in breast milk. OPO structural esters have the functions of changing intestinal microbiota, regulating lipid metabolism, promoting the absorption of calcium and fatty acids, and reducing the level of pro-inflammatory cytokines. Studies have shown that the fatty acid composition of triglycerides has different effects on lipid metabolism. Structural esters containing linoleic acid are not only easier to digest and absorb by the gastrointestinal tract than structural esters containing oleic acid, but also more conducive to the synthesis and transportation of triglycerides. However, the functional research of OPL structural esters still needs more experimental evidence. Summary of the invention

[0006] The object of the present invention is to provide an application of OPO and / or OPL in the preparation of a product for improving heat stress resistance and / or ultraviolet stress resistance. The present invention provides an application of OPL and OPO in improving the heat stress resistance and / or ultraviolet stress resistance of Caenorhabditis elegans, and the product has the effect of delaying aging or prolonging life, providing a theoretical basis for the further development, utilization and promotion of OPL and OPO, and can be used as an application in food or medicine.

[0007] Solution

[0008] In order to achieve the above objectives, in a first aspect, the present invention provides an application of OPO and / or OPL in the preparation of a product having enhanced resistance to heat stress and / or resistance to ultraviolet stress.

[0009] In a second aspect, the present invention provides a use of a preparation having OPO and OPL as active ingredients in the preparation of a product having enhanced resistance to heat stress and / or resistance to ultraviolet stress.

[0010] In a third aspect, the present invention provides a use of OPO and / or OPL in preparing a product for improving heat stress resistance and / or UV stress resistance. Optionally, the nematode is Caenorhabditis elegans.

[0011] In the first to third aspects above, further, the product is a product having the effect of delaying aging and / or prolonging life.

[0012] In the first to third aspects above, the product is a medicine, a food or a functional food.

[0013] In the first to third aspects above, the dosage form of the product is one or more of a solution, a suspension, an emulsion, a powder, a lozenge, a pill, a syrup, a tablet and a capsule.

[0014] In the first to third aspects above, the mass ratio of OPL to OPO is 1:(0-5) or 2:(0.9-3).

[0015] In the first to third aspects above, the mass ratio of OPL to OPO is 2:(0.9-2) or 2:(0.9-1.2) or 2:(0.95-1).

[0016] Beneficial Effects

[0017] The present invention provides the use of OPL and OPO in improving the heat stress resistance and / or ultraviolet stress resistance of Caenorhabditis elegans, and has the effect of delaying aging or prolonging life, providing a theoretical basis for the further development, utilization and promotion of OPL and OPO, and can be used as an application in food or medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0019] Figure 1 The survival rates of Caenorhabditis elegans grown on control NGM and NGM supplemented with different concentrations of OPL and / or OPO for 24 h and 48 h under heat stress in Test Example 1 of the present invention are shown in FIG.

[0020] Figure 2 The lifespan curves of Caenorhabditis elegans grown on control NGM and NGM supplemented with different concentrations of OPL under ultraviolet stress in Test Example 2 of the present invention are shown.

[0021] Figure 3 The lifespan curves of Caenorhabditis elegans grown on control NGM and NGM supplemented with different concentrations of OPO under ultraviolet stress in Test Example 2 of the present invention are shown.

[0022] Figure 4 The lifespan curves of Caenorhabditis elegans grown on control NGM and NGM supplemented with different concentrations of OPL and OPO under ultraviolet stress in Test Example 2 of the present invention are shown. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] In addition, in order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In some embodiments, raw materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail, so as to highlight the main purpose of the present invention.

[0025] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0026] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0027] The present invention provides the role of an OPL and / or OPO in improving the stress resistance of Caenorhabditis elegans, and provides a theoretical basis for the further development and utilization of OPL and OPO.

[0028] The anti-stress ability of Caenorhabditis elegans mainly includes resistance to heat stress and resistance to ultraviolet stress, etc., which is mainly manifested in increasing the lifespan of Caenorhabditis elegans under stress conditions and increasing the survival rate of Caenorhabditis elegans under stress stimulation in the same period of time.

[0029] In the following examples, the reagents used mainly include: M9 buffer: Dissolve 5.80 g disodium hydrogen phosphate, 3.00 g potassium dihydrogen phosphate, 0.50 g sodium chloride, and 1.00 g ammonium chloride in 1000 mL of ultrapure water. Sterilize at high temperature and high pressure and store at room temperature for later use.

[0030] Cholesterol ethanol solution: Place 1.00 g of cholesterol in 200 mL of anhydrous ethanol, wait for it to dissolve, and store at 4°C.

[0031] Magnesium sulfate solution: Dissolve 60.19 g of magnesium sulfate in 500 mL of ultrapure water, sterilize at high temperature and high pressure, and store at room temperature.

[0032] Calcium chloride solution: Dissolve 36.90 g of calcium chloride in 500 mL of ultrapure water, sterilize at high temperature and high pressure, and store at room temperature.

[0033] Phosphate buffer: Dissolve 108.30 g potassium dihydrogen phosphate and 35.60 g potassium hydrogen phosphate in 850 mL ultrapure water, sterilize at high temperature and high pressure, and store at room temperature.

[0034] Preparation of LB medium: Accurately weigh 10 g BACTO-trypsin lactone, 5 g BACTO yeast and 10 g sodium chloride, add 900 mL of ultrapure water, adjust the pH to 7.0, make up to 1000 mL, sterilize at 121°C for 20 min, and cool for use.

[0035] Cultivation and collection of OP-50 strains: Take the above 1000mL LB medium, add 100μL uracil-deficient Escherichia coli OP-50 bacterial solution (E. coli OP-50, OP-50, purchased from the Nematode Genetics Center (CGC) of the University of Minnesota), shake and culture at 37℃ and 220rpm for 24h, centrifuge at 4000rpm for 15min at 4℃, remove the supernatant, and transfer the precipitate to a centrifuge tube. Weigh the collected bacterial solution and adjust its density to 0.8g / mL. Take 200μL of the bacterial solution and spread it on the center area of ​​the NGM medium, avoiding spreading to the edge, and place it in a 37℃ incubator overnight to check whether there are any bacteria during the preparation process. The prepared OP-50 Escherichia coli is stored at 4℃.

[0036] Preparation of NGM medium: accurately weigh 4.50g sodium chloride, 37.50g agar and 3.75g BACTO-peptone, measure 1462.50mL ultrapure water, mix thoroughly, sterilize at 121℃ for 20min, add 5mg / mL cholesterol solution, 1M magnesium sulfate, 1M calcium chloride and 1M phosphate buffer after cooling and mix well. Pour the prepared NGM medium liquid into the culture dish so that the NGM completely covers the bottom of the culture dish. The volume of the medium liquid in each culture dish is constant. After drying the prepared plate overnight, store it upside down in a dry and windless place.

[0037] Example 1. Caenorhabditis elegans synchronization experiment Synchronization experiments of Caenorhabditis elegans should choose a medium suitable for the growth period of Caenorhabditis elegans, that is, a large number of eggs appear on the surface of the medium and the presence of eggs can be clearly seen in the abdomen of the adult worm. The eggs are expected to hatch into L1 larvae after 44 hours. Rinse the medium with M9 buffer, transfer the rinsed liquid to a centrifuge tube, and repeat the rinse several times to ensure that the eggs and Caenorhabditis elegans on the surface of the medium are rinsed clean. Centrifuge at 4200rpm for 10min at 4℃ and remove the supernatant. Add 20mL of M9 buffer to the precipitate and mix well. Centrifuge at 4200rpm for 10min at 4℃ and remove the supernatant, leaving about 5mL of precipitate. Slowly add an equal volume of 60% sucrose solution to the bottom of the centrifuge tube, and add 2mL of M9 buffer above the liquid. Centrifuge at 4200rpm for 6min at 4℃, aspirate the Caenorhabditis elegans and eggs in the middle layer, and transfer them to a new centrifuge tube. Centrifuge at 4200rpm for 6min at 4℃ and remove the supernatant. M9 buffer was added and mixed, and the mixture was centrifuged at 4200 rpm for 6 min at 4°C, and the supernatant was removed to remove the remaining sucrose and bacteria.

[0038] Observe the remaining Caenorhabditis elegans and egg precipitates, and add 450 μL of 5% sodium hypochlorite solution, 500 μL of 5M sodium hydroxide solution, and 4.05 mL of ultrapure water for every 0.50 mL of precipitate. Place the liquid with the lysis solution on a shaker and shake at a frequency of 200 YPM for 5 minutes for lysis. After lysis, add M9 buffer and shake the centrifuge tube vigorously to break up large debris. Centrifuge at 4200 rpm for 6 minutes at 4°C and remove the supernatant. Add M9 buffer to suspend the precipitate, filter the suspension with a 40 μL filter to filter out the unlysed worm bodies, and collect the filtrate into a new centrifuge tube. Centrifuge at 4200 rpm for 6 minutes at 4°C, remove the supernatant, and repeat the operation three times to wash the lysis solution. Pipette the suspension containing the eggs, inoculate it into a blank NGM medium without OP-50, and place it in a 20°C incubator for culture.

[0039] After 12 hours of culture, all eggs were hatched to the L1 larval stage. The surface of the culture medium was rinsed with room temperature M9 buffer, and the L1 larvae were collected in a centrifuge tube. Centrifuged at 4200 rpm for 10 minutes at 4°C, and the supernatant was removed. The M9 buffer containing L1 larvae was diluted to different multiples, and the number of Caenorhabditis elegans synchronized this time was calculated for subsequent experiments.

[0040] C. elegans culture grouping: L1-L4 stage Caenorhabditis elegans were placed in Escherichia coli OP-50 medium (NGM) containing OPL and / or OPO as a food source for Caenorhabditis elegans. Caenorhabditis elegans were synchronized by the above-mentioned synchronization experimental method before the OPL and / or OPO feeding experiment. The experiment included 1 control group and 8 experimental groups. The concentrations of OPL and / or OPO in NGM are shown in Table 1, among which groups B, C, and D were OPL feeding groups, groups E, F, and G were OPO feeding groups, and groups H and I were mixed feeding groups. After Caenorhabditis elegans were fed with OPL and / or OPO and grew to the L4 stage, subsequent experiments were carried out.

[0041] Table 1. OPL and OPO concentrations in NGM ; The life span experiment method of Caenorhabditis elegans is: After the C. elegans in the experimental and control groups grouped according to Table 1 grew to the L4 stage, approximately 200 C. elegans were transferred to NGM plates containing only E. coli OP-50 in each group. After the transfer, the worms were checked for damage, abnormal worms were excluded, the plates were inverted, placed in a biochemical incubator, incubated at 20°C, and recorded as day 0. During the first 7 days of the lifespan experiment, the C. elegans were transferred to fresh plates coated with OP-50 every 24 hours to separate the C. elegans from their offspring. Starting from the 8th day, the C. elegans were transferred to new plates every three days. At each transfer, the survival status of the C. elegans was recorded, such as survival, death, or disappearance, until all the C. elegans died. C. elegans that did not respond to mechanical stimulation were marked as dead. Remove abnormal C. elegans from the analysis, i.e., C. elegans that are still alive but exhibit abnormal survival behaviors (including hatching of live offspring inside hermaphrodites, protruding vulva, erupting vulva, paralysis or uncoordinated, bacterial or fungal contamination, etc.).

[0042] Test Example 1: Heat stress experiment After the C. elegans in the experimental group and the control group grouped according to Table 1 grew to the L4 stage, the C. elegans in each group were transferred to a 37°C incubator, placed for 2 hours, and then transferred to a 20°C incubator for recovery. The survival of C. elegans was observed 24 hours and 48 hours after recovery, and the lifespan curve of C. elegans was drawn. Each group contained about 50 C. elegans, and three independent experiments were performed.

[0043] The results are as follows Figure 1As shown in the results, supplementation with OPL and OPO can improve the survival rate of C. elegans under 37°C heat stress. Compared with the control group, the effect of improving the survival rate under heat stress in the experimental group was more significant at 48h than at 24h. The survival rate of the OPL-fed group was significantly higher than that of the OPO-fed group, but there was no significant difference between the mixed-fed groups. At 48h, the survival rate of the low-OPL group was the highest, and the survival rate of the low-OPO group was the lowest, but still higher than that of the control group. Among the mixed-fed groups, the survival rate of the OPO>OPL group was higher than that of the OPL>OPO group, which was consistent with the conclusion of the OPO or OPL-fed group alone.

[0044] Test Example 2: Ultraviolet stress experiment: UV stress experiment: After the C. elegans in the experimental group and the control group grouped as shown in Table 1 grew to the L4 stage, the C. elegans in each group were exposed to ultraviolet light for 24 h, and then transferred to an incubator at 20°C for continued cultivation. The cells were observed once a day until all the C. elegans died, and the life span curve of C. elegans was plotted.

[0045] The results are as follows Figure 2-Figure 4 As shown in the results, supplementation with OPL and OPO can extend the lifespan of C. elegans under UV stress, among which the low OPL group, medium OPO group and OPL>OPO group have significant effects on extending the lifespan of C. elegans under UV stress. The effect of extending the lifespan of C. elegans under UV stress is more obvious in the early stage of culture. Compared with the control group, the survival rate of C. elegans in the experimental group increased by 5%-35% at 5 days.

[0046] The stress resistance of Caenorhabditis elegans mainly includes resistance to heat stress and resistance to ultraviolet stress, etc. The OPL and OPO of the present invention are mainly manifested in increasing the lifespan of Caenorhabditis elegans under stress and increasing the survival rate of Caenorhabditis elegans under stress stimulation within the same period of time.

[0047] In summary, supplementation of OPL and OPO can improve the resistance of C. elegans to heat stress and UV stress.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of OPO and / or OPL in the preparation of products with enhanced resistance to heat stress and / or UV stress.

2. Application of preparations with OPO and OPL as active ingredients in the preparation of products with enhanced resistance to heat stress and / or UV stress.

3. Application of OPO and / or OPL in the preparation of products for delaying the growth of nematodes and improving their resistance to heat stress and / or UV stress.

4. The use according to claim 3, characterized in that: The nematode is Caenorhabditis elegans.

5. The use according to any one of claims 1 to 4, characterized in that: The product is a product with the effect of delaying aging and / or prolonging life.

6. The use according to any one of claims 1 to 4, characterized in that: The product is a medicine, a food or a functional food.

7. The use according to any one of claims 1 to 4, characterized in that: The dosage form of the product is one or more of solution, suspension, emulsion, powder, lozenge, pill, syrup, tablet and capsule.

8. The use according to any one of claims 1 to 4, characterized in that: The mass ratio of OPL to OPO is 1:(0~5) or 2:(0.9~3).

9. The use according to any one of claims 1 to 4, characterized in that: The mass ratio of OPL to OPO is 2:(0.9~2) or 2:(0.9~1.2).

10. The use according to any one of claims 1 to 4, characterized in that: The mass ratio of OPL and OPO is 2:(0.95~1).

Images